The initiation of an immune response against a specific antigen in mammals is brought about by the presentation of that antigen to T lymphocytes. An antigen is presented to T lymphocytes in the context of a major histocompatability (MHC) complex (also referred to as HLA in humans and H-2 in mice). The three-dimensional structure of the MHC includes a groove or cleft into which the presented antigen fits. When an appropriate receptor on a T lymphocyte (also known as the T cell receptor, or TCR) interacts with the MHC/antigen complex on an antigen-presenting cells (APC) in the presence of necessary co-stimulatory signals, the T lymphocyte is stimulated, triggering various aspects of the well characterized cascade of immune system activation events, including induction of cytotoxic T lymphocyte (CTL) function, induction of B lymphocyte function and stimulation of cytokine production (see, e.g. Roitt, I and Delves, P. Roitt's Essential Immunology, 10th Ed., Boston, Blackwell Science, 2002; Abbas, A. et al. Cellular and Molecular Immunology, W.B. Saunders Company, Philadelphia, 1991; Silverstein, A. A History of Immunology, San Diego, Academic Press, 1989).
MHC/peptide complexes have a low affinity for the TCR, and dissociate rapidly with a t1/2 of approximately 2 to 12 seconds (measured at 25° C.) (see, e.g., Matsui, K. et al. (1994) Proc. Natl. Acad. Sci., USA 91:12862). Multimerization on a steptavidin scaffold permits multivalent binding to the TCR and slow dissociation of tetramers from T cells with the appropriate MHC/peptide specificity (i.e., t1/2 of 20 to 240 minutes for tetramer dissociation from murine cytochrome C specific T cells) (see, e.g., Savage, P. A. et al. (1999) Immunity 10:485). The ability to directly visualize antigen specific CD8+T cells has led to a reappraisal of the magnitude of virus specific T cell responses in acute and chronic infections since the frequency of such T cells had been greatly underestimated by limiting dilution analyses (Murali-Krishna, K. et al. (1998) Immunity 8:177). Tetramers for MHC class I/peptide complexes have therefore become an indispensable tool for both basic and clinical scientists in the investigation of anti-viral T cell responses. The success of tetramers of MHC class I/peptide complexes is largely due to the fact that the MHC class I heavy chain and β2-microglobulin can be reliably refolded from subunits expressed in E. coli in the presence of appropriate peptides (see, e.g., Garboczi, D. N. et al. (1992) Proc. Natl. Acad. Sci., USA 89:3429 and Altman, J. D. et al. (1996) Science 274:94).
By comparison, development of MHC class II tetramers has been far more challenging. Thus far, antigen-specific human CD4+ T cells have only been detected with MHC class II tetramers in a single human disease, chronic Lyme arthritis, where T cells against a previously defined peptide from the OspA antigen were visualized in the synovial fluid from two of three patients with the disease (Meyer, A. L. et al. (2000) Proc. Natl. Acad. Sci., USA 97:11433). The difficulty in the application of MHC class II tetramers to the investigation of human diseases relates, at least in part, to the greater difficulty of expressing functional molecules. The majority of human and murine MHC class II molecules have not been successfully refolded from α and β chains produced in E. coli, which has necessitated the development of alternative approaches (see, e.g., Frayser, M. et al. (1999) Protein Expr. Purif. 15:105). Expression of the MHC α and β ectodomains in eukaryotic cells results in the secretion of “empty” molecules, and a fraction of these molecules can be loaded with peptides (see, e.g., Stern L. J. and Wiley, D. C. (1992) Cell 68:465). Depending on the isotype and allele, such “empty” molecules have a tendency to aggregate and a substantial fraction of these molecules can be inactive. Tetramers have been made using this approach and have been shown to stain T cell lines grown in vitro with viral antigen-peptide in the presence of recombinant interleukin (IL)-2, but have not yet been used to directly identify virus-specific T cell populations from peripheral blood without prior in vitro expansion of T cells (see, e.g., Novak, E. J. et al. (1999) J. Clin. Invest. 104:R63 and Kwok, W. W. et al. (2000) J. Immunol. 164:4244). Reasons for these shortcomings include very low frequency of virus specific T cells and/or insufficient binding of tetramers to relevant T cell populations.
Due to the long half-life of bound peptides, the kinetics of peptide binding to HLA-DR molecules purified from antigen presenting cells is very slow. Even with extended incubation times, only a small fraction of MHC class II molecules can be loaded with defined peptides since high affinity peptides have exceptionally long half-lives (see, e.g., Lanzavecchia, A. et al. (1992) Nature 357:249 and Jensen, P. E. et al. (1992) J. Exp. Med. 176:793). The kinetics of peptide association are also relatively slow for empty DR1 molecules refolded from subunits expressed in E. coli in the absence of any peptide. Biphasic kinetics were observed with τ=4 hours for the initial faster phase and τ=24 hours for the second slower phase, and even after an extended incubation (4 days) only a fraction of molecules were loaded with the high affinity influenza HA peptide. The slow kinetics are due to the fact that only a small fraction of empty DR1 molecules (1-5%) are in a peptide-receptive form, and that the overall reaction rate is determined by the slow conversion from the peptide-averse form (Zarutskie, J. A. et al. (1998) J. Exp. Med. 188:2205).
To solve this problem, MHC class II tetramers have been generated by covalently linking the peptide of interest to the N-terminus of the MHC class II β chain (see, e.g., Kappler, J. W. and Marrack, P., U.S. Pat. No. 5,820,866; Crawford, F. et al. (1998) Immunity 8:675; and Kozono, H. et al. (1994) Nature 369:151). While this approach is particularly suitable when a single tetramer is required (i.e., such as for tracking T cells from TCR transgenic mice), it is not suited to a clinical setting where the relevant T cell epitopes are not yet known with certainty because of the need to create a new transfectant/recombinant virus for each peptide of interest. In addition, due to the variable contributions of a number of peptide residues (at least 4 or 5) involved in MHC class II binding, epitope prediction methods for MHC class II molecules are not as reliable as those widely used for MHC class I molecules, even though these prediction methods are based on detailed biochemical studies with several MHC class II molecules (see, e.g., Hammer, J. et al. (1993) Cell 74:197). As such, successful application of MHC class II compounds, such as MHC class II tetramers, to a clinical setting would require an approach that permits the generation of sets of compounds with biochemically well-defined MHC class II/peptide complexes, preferably such that many different peptides can be evaluated without prior knowledge of relevant CD4+ T cell epitopes.